The most highly conserved regions of proteins can be represented as blocks of aligned sequence segments, typically with multiple blocks for a given protein family. The investigators have developed and validated improved methodologies for multiple alignment comparison: position-based sequence weights efficiently neutralize sequence redundancy in a simple and intuitive way, position-based pseudo-counts greatly improve searching performance over current widely used methods for column representation, blocks-versus-blocks searching allows sensitive detection of subtle relationships, and embedding methods effectively deal with problems caused by non-conserved regions between blocks. In addition, the investigators expanded the Blocks WWW and e-mail sites by adding Block Maker for making blocks from user-defined families, LAMA for searching the Blocks Database with user-generated blocks, COBBLER for automated sequence database searching of consensus-embedded sequences, sequence logo displays of blocks, and automated tree-making from multi-block alignments. Furthermore, the Blocks Database has been expanded by merging automatically generated Prosite-based blocks with manually generated Print blocks for purposes of searching and documentation retrieval. The investigators have begun to exploit these powerful new tools to address biological questions of interest in the laboratory, including characterization of novel DNA methyltransferases and inteins. They propose to continue expansion of their Internet site by improving methodologies and adding new capabilities, and to exploit these new biological discoveries experimentally. One new capability that they propose is full implementation of a new strategy for degenerate PCR primer design and its experimental verification using C(5) DNA methyltransferases. This effort could yield new eukaryotic family members of great importance for understanding epigenetic phenomena, some of which, such as parental imprinting, are relevant to human disease.